Human Embryo Kidney Cells Research Articles

Synthesis, characterization, and biological evaluation of some metal complexes containing N and S donor atoms

Eight new bipyridine adducts of Zn(II) and Ni(II) dithiocarbamate complexes have been prepared from the reaction of Zn(II) and Ni(II) complexes of N-ethyl-N-phenyldithiocarbamate and 4,4′ dipyridine, 4,4′-dimethyl-2,2′-bipyridine, 4,4′-dimethoxy-2,2′-bipyridine, 4,4′-ditertbutyl-2,2′-bipyridine. The resulting adducts were represented as [M(L1)2(DP)], [M(L1)2(DMeB)], [M(L1)2(DMxB)] and [M(L1)2(DTB)] respectively (where M = Zn or Ni). The structural configuration of these adducts was established using various spectroscopic techniques. The obtained data from the spectroscopic studies revealed a change in the tetrahedral configuration of the parent complexes to octahedral coordination in the adducts due to an additional metal-nitrogen bond. The formation of this bond was established by a considerable shift in the peaks around the metal-nitrogen (M−N) coordination upon the formation of the adducts. The study of biological properties including cytotoxicity, antioxidant, and anti-inflammatory properties were carried out. No noticeable trends were reported in all the studies despite the concentration-dependent profile shown in the assays and the structural similarities of the compounds. These adducts, nevertheless, showed good antioxidant activity in the DPPH assay, with IC50 ranging between 3.78 and 4.87 µg/mL which is better than the standard ascorbic acid (4.96 µg/mL). Also, in the cytotoxicity study, the adducts [Zn(L)2DMeBp] (1) and [Ni(L)2 DTBp] (6) showed the best activity against the mutating human cervical cancer (HeLa) and embryo kidney (HEK 293) cell lines and better than the standard 5-Flurouracil. The molecular docking studies further revealed that the adducts had a good binding affinity to the drug targets used for the study.

Kei-apple-mediated NiO nanoparticles and biological studies: anti-inflammatory and cytotoxicity study against HeLa and HEK 293 cell lines

Nickel oxide (NiO) nanoparticles are gaining popularity in multiple fields owing to their useful properties. The application in biomedicine has been further enhanced by combining them with plant extracts that possess unique biological properties. In this report, NiO nanoparticles were synthesized by mediating the reaction process with the leaf extracts of Kei-apple (Dovyallis Caffra), a local fruit tree found in South Africa. Various characterization techniques such as x-ray diffraction (XRD), V–Vis spectroscopy (UV–Vis), transmission electron microscope (TEM), Scanning electron microscope (SEM), and Energy dispersive x-ray analysis (EDX) were employed to establish the different physicochemical properties of the prepared NiO material. The XRD report obtained confirmed the formation of NiO nanoparticles with an average crystallite size of 9 mm. Furthermore, the microscopic techniques showed that the material possessed a spherical-shaped structure, with diameter sizes smaller than 27.18 nm (average size of about 11 nm). The purity of the material was shown in the EDX analysis, in which the primary composition was only the elemental constituents of the NiO nanoparticles. The nanoparticles exhibited good cytotoxicity comparable to the 5-Fluorouracil in both the Human embryo kidney (HEK 293) and Human cervical cancer (HeLa) cell lines, with a half maximal inhibitory concentration (IC50) value of 0.00015 and 13.8 μg ml−1, respectively. The anti-inflammatory study, on the other hand, exhibited a weak anti-inflammatory effect in the used Bovine serum albumin denaturation assay. The finding here thus suggests that the Kei-apple mediated NiO nanoparticles can be safely used in different fields without causing any appreciable harm to the human body due to the specificity to the cancerous cell line and the observed weak viability in the used non-cancerous embryonic kidney cells.

Indolyl-Derived 4H-Imidazoles: PASE Synthesis, Molecular Docking and In Vitro Cytotoxicity Assay

The strategy of the nucleophilic substitution of hydrogen (SNH) was first applied for the metal-free C-H/C-H coupling reactions of 4H-imidazole 3-oxides with indoles. As a result, a series of novel bifunctional azaheterocyclic derivatives were obtained in yields up to 95%. In silico experiments on the molecular docking were performed to evaluate the binding possibility of the synthesized small azaheterocyclic molecules to the selected biotargets (BACE1, BChE, CK1δ, AChE) associated with the pathogenesis of neurodegenerative diseases. To assess the cytotoxicity for the synthesized compounds, a series of in vitro experiments were also carried out on healthy human embryo kidney cells (HEK-293). The leading compound bearing both 5-phenyl-4H-imidazole and 1-methyl-1H-indole moieties was defined as the prospective molecule possessing the lowest cytotoxicity (IC50 > 300 µM on HEK-293) and the highest binding energy in the protein–ligand complex (AChE, −13.57 kcal/mol). The developed compounds could be of particular interest in medicinal chemistry, particularly in the targeted design of small-molecule candidates for the treatment of neurodegenerative disorders.

Towards Autonomous Process Control—Digital Twin for HIV-Gag VLP Production in HEK293 Cells Using a Dynamic Metabolic Model

Despite intensive research over the last three decades, it has not yet been possible to bring an effective vaccine against human immunodeficiency virus (HIV) and the resulting acquired immunodeficiency syndrome (AIDS) to market. Virus-like particles (VLP) are a promising approach for efficient and effective vaccination and could play an important role in the fight against HIV. For example, HEK293 (human embryo kidney) cells can be used to produce virus-like particles. In this context, given the quality-by-design (QbD) concept for manufacturing, a digital twin is of great importance for the production of HIV-Gag-formed VLPs. In this work, a dynamic metabolic model for the production of HIV-Gag VLPs was developed and validated. The model can represent the VLP production as well as the consumption or formation of all important substrates and metabolites. Thus, in combination with already described process analytical technology (PAT) methods, the final step towards the implementation of a digital twin for process development and design, as well as process automation, was completed.

Ultrastructural Features of Gold Nanoparticles Interaction with HepG2 and HEK293 Cells in Monolayer and Spheroids.

Use of multicellular spheroids in studies of nanoparticles (NPs) has increased in the last decade, however details of NPs interaction with spheroids are poorly known. We synthesized AuNPs (12.0 ± 0.1 nm in diameter, transmission electron microscopy (TEM data) and covered them with bovine serum albumin (BSA) and polyethyleneimine (PEI). Values of hydrodynamic diameter were 17.4 ± 0.4; 35.9 ± 0.5 and ±125.9 ± 2.8 nm for AuNPs, AuBSA-NPs and AuPEI-NPs, and Z-potential (net charge) values were −33.6 ± 2.0; −35.7 ± 1.8 and 39.9 ± 1.3 mV, respectively. Spheroids of human hepatocarcinoma (HepG2) and human embryo kidney (HEK293) cells (Corning ® spheroid microplates CLS4515-5EA), and monolayers of these cell lines were incubated with all NPs for 15 min–4 h, and fixed in 4% paraformaldehyde solution. Samples were examined using transmission and scanning electron microscopy. HepG2 and HEK2893 spheroids showed tissue-specific features and contacted with culture medium by basal plasma membrane of the cells. HepG2 cells both in monolayer and spheroids did not uptake of the AuNPs, while AuBSA-NPs and AuPEI-NPs readily penetrated these cells. All studied NPs penetrated HEK293 cells in both monolayer and spheroids. Thus, two different cell cultures maintained a type of the interaction with NPs in monolayer and spheroid forms, which not depended on NPs Z-potential and size.

Co-administration of vitamin E and selenium in vivo and in vitro ameliorates the toxic effects caused by ivermection and doramectin

Avermectins are used in animals and humans for their broad-spectrum effects against parasites causing cytotoxicity and damage to the cellular DNA. In this study, we examined the toxicological changes of ivermectin (IVM) and doramectin (DME) with or without the co-administration of vitamin E (Vit. E) and selenium (Se). The drugs used were for animal use. Twenty-five adult male rats were divided into five groups. Group 1 (control) was given saline, Group 2 was given IVM (0.2 mg/kg b.w.), Group 3 was given IVM and Vit. E/Se (80/1.6 mg/kg b.w., respectively), Group 4 received DME (0.2 mg/kg b.w.), and Group 5 received DME and Vitamin E/Se. Both IVM and DME were given by subcutaneous injections whereas Vit. E and Se were given orally. All the treatments were given once per week throughout the eight weeks. Although the doses were off-label use, they were given in a long-term course to unveil their toxicity effects in a clear manner and the response of the amelioration. By 24-h after the 8<sup>th</sup> week, the rats were sacrificed. Their blood was sampled for the haematological and serobiochemical examinations. Histopathological changes and caspase-3 were determined in the hepatic and renal tissues. The histopathological findings showed that Vit. E and Se reduced the cellular changes induced by IVM or DME, indicating that Vit. E and Se protect against both types of avermectins, and that DME was safer than IVM. The cytotoxicity was assessed on a human embryo kidney (HEK) and skin cells by the SRB/IC<sub>50</sub> method and AO/EB (acridine orange-ethidium bromide) staining. Both IVM and DME caused apoptosis in the cultured HEK more than in the skin cells (80% vs. 30%, respectively). The cellular apoptosis in response to the IVM was more than that of DME, and the use of Vit. E and Se reduced the cytotoxicity as observed by caspase-3, in vivo, and IC<sub>50</sub>, in vitro.

Specificity and nanomolar potency of melatonin on G-protein coupled melatonin MT1 and MT2 receptors expressed in HEK-293T human embryo kidney cells

This is a pre-registered study, i.e. a study whose hypotheses and experiments designed to address these hypotheses have been deposited in a database before starting the experiments. The study aims at assessing the Gs versus Gi coupling and the potency of melatonin in the human version of melatonin MT1 and MT2 G-protein-coupled receptors expressed in HEK-293T cells. The results show that these receptors are Gi but not Gs coupled. By using a standard procedure of modulation of 0.5 µM forskolin-induced cAMP levels, it was found that the potency on MT2 receptor-mediated actions is in the low nanomolar range, but the potency on MT1 receptor is in the high nanomolar range. The potency of melatonin to stimulate the MT2 receptor is similar to that of a selective agonist, N-[2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethyl]butanamide (IIK7). Overall, the data on the potency of melatonin on its receptors will provide a new look for melatonin research. It is important to consider this finding for appropriately addressing physiological or therapeutic effects based on melatonin potency. Thus, the low doses of melatonin used in the existing prolonged release preparations or in other supplements should be revisited.

Essential role of glucokinase in the protection of pancreatic \u03b2 cells to the glucose energetic status

Energy sensing is indispensable to balance anabolic and catabolic processes for the maintenance of cell viability. Pancreatic β cells are especially relevant because of their involvement in the coordination of insulin secretion when glucose concentration arises in the local milieu. In this work, we uncover the increased susceptibility of pancreatic β cells to cell death in response to different energy stressors. Upon glucose decline, from 25 to 5 mM, caused by stimulation with either 2-deoxyglucose or metformin, only pancreatic β cells showed an increase in cell death. Very interestingly, when we transfected either mouse insulinoma cell or human embryo kidney cells with a phospho-mutant form of B cell lymphoma 2 associated agonist of cell death at serine 155 (BAD S155D), an increase in the pro-survival factor B cell lymphoma 2 was detected in pancreatic β cells and not in human embryonic kidney cells in the presence of the energetic stressors. This data suggests that the protective capacity of this mutant form is only present in cells that present glucokinase. In contrast, upon hyperactivation of mechanistic target of rapamycin complex 1 signaling by knocking-down tuberous sclerosis complex protein, we observed increased susceptibility to cell death in response to energy stress in both pancreatic and non-pancreatic β cells. Therefore, mechanistic target of rapamycin complex 1 signaling presents a dual effect on cell viability. On the one hand, a chronic inhibition of mechanistic target of rapamycin complex 1 activity in response to the energy status is deleterious for pancreatic β cells, being attenuated by the overexpression of B cell lymphoma 2 associated agonist of cell death S155D. On the other hand, mechanistic target of rapamycin complex 1 hyperactivity provokes a susceptibility to energetic stress-induced cell death. Taken together, these results may open potential implications for the use of glucokinase activators or mechanistic target of rapamycin complex 1 modulators for the maintenance of pancreatic β cells for longer periods of time avoiding its loss in different pathologies such as type 2 diabetes mellitus.

Sequence optimization and glycosylation of vasoinhibin: Pitfalls of recombinant production

Vasoinhibin belongs to a family of proteins with antiangiogenic properties derived by proteolytic cleavage from the hormone prolactin (PRL). Vasoinhibin isoforms range from the first 79 to the first 159 residues of PRL. In an attempt to increase the yield of recombinant vasoinhibin and avoid incorrect intra- and inter-disulfide bond formation, the cDNA sequence comprising the first 123 amino acids of human PRL, in which cysteine 58 was or not mutated to serine, was codon-optimized. The optimized constructs achieved a 6-fold increase in mRNA expression but showed no change in protein production and reduced protein secretion when expressed in human embryo kidney (HEK293T/17) cells. Limited vasoinhibin levels associated with the activation of the unfolded protein response (UPR) and endoplasmic reticulum-associated degradation (ERAD) as revealed by the upregulation of UPR (Bip, Xbp-1, and Chop) and ERAD (Hrd1, Os9, and Sel1l) target genes. Mutation to serine introduced a new N-glycosylation site and associated with increased glycosylation and release of glycosylated vasoinhibin isoforms having reduced antiangiogenic properties. We conclude that overexpression and excessive glycosylation lead to protein degradation and reduced bioactivity, respectively, negatively affecting the production of recombinant vasoinhibin in mammalian cells.

Immortalization of human umbilical vein endothelial cells and the effect of immortalization on its biological characteristics

Objective To establish the immortalized human umbilical vein endothelial cell lines and investigate the effect of immortalization on biological characteristics of proliferation, apoptosis and tumorigenesis of the cells. Methods Plasmid containing simian virus 40 large T antigen (SV40LT) gene or human telomerase reverse transcriptase (hTERT) gene was amplified and extracted; the target genes marked by puromycin resistance were packed by human embryo kidney cells (293T) to get the retroviral vectors and used to infect human umbilical vein endothelial cells (HUVECs); then the infected cells were screened by culture medium containing 2 μg/ml puromycin for two weeks; and polymerase chain reaction (PCR) was used to identify whether the target genes were successfully transferred into HUVECs; cell counting method, flow cytometry and tumorigenesis experiments in nude mice were used to detect the biological characteristics of proliferation, apoptosis and malignant transformation of immortalized cells respectively. Results The human umbilical vein endothelial cells were successfully immortalized by retrovirus-mediated SV40LT and hTERT; On the 5th to the 7th day, there were significant differences in cells number between the SV40LT immortalized group and HUVECs group (P<0.05) and between the hTERT immortalized group and HUVECs group (P<0.05). The apoptosis rate of HUVECs group was (11.2±0.9)%, the apoptosis rate of HUVECs immortalized by SV40LT was (3.4±0.4)%, the apoptosis rate of HUVECs immortalized by hTERT was (2.8±0.4)%. The apoptotic rates of the two immortalized group were lower than that of HUVECs group (both P<0.01). These immortalized HUVECs had no tumorigenicity. Conclusion Retrovirus-mediated SV40LT and hTERT can successfully immortalize HUVECs; the proliferative capacity of immortalized HUVECs is greatly enhanced; the apoptosis rate is decreased, and no malignant transformation occurs. Key words: Simian virus 40 large T antigen; Human telomerase reverse transcriptase; Retroviral vector; Human umbilical vein endothelial cell; Immortalization

Establishment and Validation of an In Vitro Screening Method for Traditional Chinese Medicine-Induced Nephrotoxicity.

Renal injury is among the adverse drug reactions (ADRs) caused by herbal medicine products (HMPs). Traditional Chinese medicines (TCMs) have been practiced for over 2000 years in China and East Asia, and herbs are currently used worldwide for the treatment and prevention of chronic and acute disease. Operetta high content analysis (HCA, PerkinElmer, Waltham, MA, USA), which is an in vitro, sensitive, reproducible, multiparametric screening method, was used to evaluate the cytotoxicity of HMPs in cultures of HEK293 human embryo kidney cells. Cytotoxic results were validated by an animal-based subacute toxicity assay. The renal safety of 18 active pharmaceutical agents from 13 TCM herbs with known nephrotoxic potential was evaluated in HEK293 human embryonic kidney cells. A panel of five parameters, cell viability, nuclear area, nuclear roundness, mitochondrial mass, and mitochondrial membrane potential, was utilized to evaluate drug-induced renal mitochondrial and nuclear injury. HCA can be a useful tool for preclinical screening and postclinical evaluation of HMPs. The nephrotoxicity of diosbulbin B and other HMPs was evident at a concentration as low as 0.01 μM.

Comparison of cell-based versus cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor.

The human bone morphogenetic protein-2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell-free synthesis in CHO cell lysates containing microsomal structures as sites of post-translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153pg/mL. Up to 280ng/mL could be produced in the transient expression system. In both cases the rhBMP-2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell-free system, hBMP2 yields could be increased to almost 40μg/mL, reached within three hours. The cell-free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post-translational processing.

Inhibitory Effect of Abitylguanide on Adenovirus Replication.

N'N'-anhydro-bis(β-hydroxy-ethyl)biguanide.HCl (abitylguanide) demonstrated a marked inhibitory effect on replication in cell cultures of a broad spectrum of human adenoviruses both standard laboratory strains and strains isolated from epidemic keratoconjunctivitis patients. The strongest inhibitory activity was found in viruses belonging to subgroup C (Rosen's subgroup III). The compound susceptible period of human adenovirus 5 replication in primary cell cultures of human embryo kidney cells included the total replication cycle and was especially pronounced during the exponential phase of the virus growth curve. Electron microscopy established that the compound decreased the percentage of cells in which mature or empty virions with the characteristic nuclear localization were observed; a complete absence of paracrystals was registered and the number of cells with virus particles arranged in crystals in the nucleoplasm was strongly decreased. Abitylguanide can be considered as a ligand of adenovirus capsid protein(s).

A Biodegradable Polycationic Paint that Kills Bacteria in Vitro and in Vivo.

Bacterial colonization and subsequent formation of biofilms onto surfaces of medical devices and implants is a major source of nosocomial infections. Most antibacterial coatings to combat infections are either metal-based or nondegradable-polymer-based and hence limited by their nondegradability and unpredictable toxicity. Moreover, to combat infections effectively, the coatings are required to display simultaneous antibacterial and antibiofilm activity. Herein we report biocompatible and biodegradable coatings based on organo-soluble quaternary chitin polymers which were immobilized noncovalently onto surfaces as bactericidal paint. The polycationic paint was found to be active against both drug-sensitive and -resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant Klebsiella pneumoniae. The cationic polymers were shown to interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thereby causing leakage of intracellular constituents and cell death upon contact. Importantly, surfaces coated with the polymers inhibited formation of biofilms against both Gram-positive S. aureus and Gram-negative E. coli, two of the most clinically important bacteria that form biofilms. Surfaces coated with the polymers displayed negligible toxicity against human erythrocytes and embryo kidney cells. Notably, the polymers were shown to be susceptible toward lysozyme. Furthermore, subcutaneous implantation of polymer discs in rats led to 15-20% degradation in 4 weeks thereby displaying their biodegradability. In a murine model of subcutaneous infection, polymer-coated medical-grade catheter reduced MRSA burden by 3.7 log compared to that of noncoated catheter. Furthermore, no biofilm development was observed on the coated catheters under in vivo conditions. The polycationic materials thus developed herein represent a novel class of safe and effective coating agents for the prevention of device-associated infections.

Antiproliferative Activity of Niclosamide Against Melanoma and Colorectal Cancer Cells

Studies of the actions of niclosamide against tumor cells and normal human cells (human embryo kidney cells and pulmonary embryo fibroblast cells) showed higher levels of cytotoxic activity against tumor cells, including melanoma (the Mel-8, Mel-10, MS lines) and colorectal cancer (the Caco-2, COLO 320 HSR, and SW827 cells). Niclosamide was shown to have a high level of cytotoxic activity against cells with the multidrug resistance phenotype due to high levels of expression of MDR1 protein on the plasma membrane (the Mel-8 and MCF-7Adr lines). The proportion of tumor stem cells (TSC) was measured by flow cytometry as the proportion of cells forming side populations (SP). Niclosamide was found to act on TSC cells either more effectively (colorectal cancer line SW 837, (decreases in SP fraction size) or with the same effectiveness as the main population of tumor cells (SP fraction size unaltered).

Chitosan Derivatives Active against Multidrug-Resistant Bacteria and Pathogenic Fungi: In Vivo Evaluation as Topical Antimicrobials.

The continuous rise of antimicrobial resistance and the dearth of new antibiotics in the clinical pipeline raise an urgent call for the development of potent antimicrobial agents. Cationic chitosan derivatives, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chlorides (HTCC), have been widely studied as potent antibacterial agents. However, their systemic structure-activity relationship, activity toward drug-resistant bacteria and fungi, and mode of action are very rare. Moreover, toxicity and efficacy of these polymers under in vivo conditions are yet to be established. Herein, we investigated antibacterial and antifungal efficacies of the HTCC polymers against multidrug resistant bacteria including clinical isolates and pathogenic fungi, studied their mechanism of action, and evaluated cytotoxic and antimicrobial activities in vitro and in vivo. The polymers were found to be active against both bacteria and fungi (MIC = 125-250 μg/mL) and displayed rapid microbicidal kinetics, killing pathogens within 60-120 min. Moreover, the polymers were shown to target both bacterial and fungal cell membrane leading to membrane disruption and found to be effective in hindering bacterial resistance development. Importantly, very low toxicity toward human erythrocytes (HC50 = >10000 μg/mL) and embryo kidney cells were observed for the cationic polymers in vitro. Further, no inflammation toward skin tissue was observed in vivo for the most active polymer even at 200 mg/kg when applied on the mice skin. In a murine model of superficial skin infection, the polymer showed significant reduction of methicillin-resistant Staphylococcus aureus (MRSA) burden (3.2 log MRSA reduction at 100 mg/kg) with no to minimal inflammation. Taken together, these selectively active polymers show promise to be used as potent antimicrobial agents in topical and other infections.

Effect of Thermodiffusion Nitriding on Cytocompatibility of Ti-6Al-4V Titanium Alloy

The nitrided layer was formed on the surface of Ti-6Al-4V titanium alloy by the thermodiffusion saturation in nitrogen at the atmospheric pressure. The study of the vitality of pseudonormal human embryo kidney cells of the HEK293T line showed that their cultivation in the presence of the untreated alloy sample is accompanied by a statistically significant reduction in the number of living cells compared with the control sample (untreated cells), whereas their cultivation in the presence of the nitrided alloy sample does not change the cell number considerably. In addition, it was shown that cell behavior in the presence of the nitrided sample differs only slightly from the control sample, whereas the growth of cells in the presence of the untreated alloy differed significantly from that in the control sample, demonstrating small groups of cells instead of their big clusters.

Effect of Poly(ethylene glycol) Spacer on Peptide-Decorated Hepatocellular Carcinoma-Targeted Lipoplexes In Vitro.

Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths and is particularly prevalent in regions where Hepatitis B is highly endemic. Classical treatment options are largely limited to surgical measures and transcatheter chemoembolization. However biopanning of phage display libraries has identified very selective HCC-targeting peptides, which may be tethered to cationic liposomes for non-viral delivery of therapeutic DNA to affected cells thus providing a foundation for the development of new gene therapy treatment approaches. In this investigation, a cysteinylated HCC-tropic peptide has been linked directly to cationic liposomes, containing the cytofectin 3β-[N-(N',N'-dimethlaminopropylamino)-carbamoyl]-cholestero (Chol-T) and dioleolphosphatidyl ethanolamine, via a novel membrane-embedded maleimido cholesteryl derivative 3-[N-(hydrazino-y-maleimidobutyryl)-carbamoyl] cholesterol (Chol-Mal), or with an intervening poly(ethylene glycol) spacer element. The effect that inclusion of the hydrophilic polymer has on targeted lipoplex-mediated gene delivery in the human HCC cell line HepG2 has been investigated. Results show that both systems bind DNA and transfect this cell line with equal efficiency, while transgene expression levels in human embryo kidney cells HEK293 were low and comparable to those achieved in competition assays in HepG2 cells and by lipoplexes decorated with scrambled peptides. Although inclusion of poly(ethylene glycol) in formulations should be considered for in vivo studies, we show here that it offers no advantage in this application in vitro.

Polymerizable disulfide paclitaxel prodrug for controlled drug delivery

A polymerizable disulfide paclitaxel (PTX) prodrug was synthesized by the consequential esterification reactions of 3,3′-dithiodipropionic acid (DTPA), a disulfide compound containing two active carboxyl groups, with 2-hydroxyethyl methacrylate (HEMA) and PTX. The structure of the prodrug was confirmed by 1H NMR characterization. Then, the polymerizable prodrug was copolymerized with poly(ethylene glycol) methyl ether methacrylate (PEGMEA) to obtain a copolymer with hydrophilic PEG side chains and PTX covalently linked onto the backbone via disulfide bonds. The loading content of PTX was 23%. In aqueous solution, this copolymer prodrug could self-assemble into micelles, with hydrophobic PTX as the cores and hydrophilic PEG-segment as the shells. In vitro cell assay demonstrated that this copolymer prodrug showed more apparent cytotoxicity to cancer cells than to human normal cells. After incubation for 48h, the cell viability of HEK-293 cells (human embryo kidney cells) at 0.1μg/mL PTX still remained more than 90%, however, that of HeLa cells (human cervical cancer cells) decreased to 52%.

Induction of cytopathogenicity in human glioblastoma cells by chikungunya virus.

Chikungunya virus (CHIKV), an arthritogenic old-world alphavirus, has been implicated in the central nervous system (CNS) infection in infants and elderly patients. Astrocytes are the major immune cells of the brain parenchyma that mediate inflammation. In the present study we found that a local isolate of CHIKV infect and activate U-87 MG cells, a glioblastoma cell line of human astrocyte origin. The infection kinetics were similar in infected U-87 MG cells and the human embryo kidney (HEK293) cells as indicated by immunofluorescence and plaque assays, 24h post-infection (p.i.). In infected U-87 MG cells, apoptosis was detectable from 48h p.i. evidenced by DNA fragmentation, PARP cleavage, loss of mitochondrial membrane potential, nuclear condensation and visible cytopathic effects in a dose and time-dependent manner. XBP1 mRNA splicing and eIF2α phosphorylation studies indicated the occurrence of endoplasmic reticulum stress in infected cells. In U-87 MG cells stably expressing a green fluorescent protein-tagged light chain-3 (GFP-LC3) protein, CHIKV infection showed increased autophagy response. The infection led to an enhanced expression of the mRNA transcripts of the pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and CXCL9 within 24h p.i. Significant up-regulation of the proteins of RIG-I like receptor (RLR) pathway, such as RIG-I and TRAF-6, was observed indicating the activation of the cytoplasmic-cellular innate immune response. The overall results show that the U-87 MG cell line is a potential in vitro model for in depth study of these molecular pathways in response to CHIKV infection. The responses in these cells of CNS origin, which are inherently defective in Type I interferon response, could be analogous to that occurring in infants and very old patients who also have a compromised interferon-response. The results also point to the intriguing possibility of using this virus for studies to develop oncolytic virus therapy approaches against glioblastoma, a highly aggressive malignancy.